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Biochemistry

Measurement of FNR-NrdI Interaction by Microscale Thermophoresis (MST) Authors:  Ingvild Gudim, Marie Lofstad, Marta Hammerstad and Hans-Petter Hersleth, date: 04/20/2017, view: 3061, Q&A: 0
This protocol describes how to measure protein-protein interactions by microscale thermophoresis (MST) using the MonolithTM NT.115 instrument (NanoTemper). We have used the protocol to determine the binding affinities between three different flavodoxin reductases (FNRs) and a flavodoxin-like protein, NrdI, from Bacillus cereus ...
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Determination of Hydrodynamic Radius of Proteins by Size Exclusion Chromatography Authors:  Valentina La Verde, Paola Dominici and Alessandra Astegno, date: 04/20/2017, view: 6030, Q&A: 0
Size exclusion chromatography (SEC) or gel filtration is a hydrodynamic technique that separates molecules in solution as a function of their size and shape. In the case of proteins, the hydrodynamic value that can be experimentally derived is the Stokes radius (Rs), which is the radius of a sphere with the same hydrodynamic properties ( ...
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Expression, Purification and Crystallisation of the Adenosine A2A Receptor Bound to an Engineered Mini G Protein Authors:  Byron Carpenter and Christopher G. Tate, date: 04/20/2017, view: 3111, Q&A: 0
G protein-coupled receptors (GPCRs) promote cytoplasmic signalling by activating heterotrimeric G proteins in response to extracellular stimuli such as light, hormones and nucleosides. Structure determination of GPCR–G protein complexes is central to understanding the precise mechanism of signal transduction. However, these complexes are ...
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Expression and Purification of Mini G Proteins from Escherichia coli Authors:  Byron Carpenter and Christopher G. Tate, date: 04/20/2017, view: 3230, Q&A: 0
Heterotrimeric G proteins modulate intracellular signalling by transducing information from cell surface G protein-coupled receptors (GPCRs) to cytoplasmic effector proteins. Structural and functional characterisation of GPCR–G protein complexes is important to fully decipher the mechanism of signal transduction. However, native G proteins are ...
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Affinity Purification of the RNA Degradation Complex, the Exosome, from HEK-293 Cells Authors:  Michal Domanski and John LaCava, date: 04/20/2017, view: 2195, Q&A: 0
The RNA exosome complex plays a central role in RNA processing and regulated turnover. Present both in cytoplasm and nucleus, the exosome functions through associations with ribonucleases and various adapter proteins (reviewed in [Kilchert et al., 2016]). The following protocol describes an approach to purify RNA exosome complexes from ...
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Cancer Biology

Melanoma Stem Cell Sphere Formation Assay Authors:  Alessandra Tuccitto, Valeria Beretta, Francesca Rini, Chiara Castelli and Michela Perego, date: 04/20/2017, view: 2937, Q&A: 0
Self-renewal is the ability of cells to replicate themselves at every cell cycle. Throughout self-renewal in normal tissue homeostasis, stem cell number is maintained constant throughout life. Cancer stem cells (CSCs) share this ability with normal tissue stem cells and the sphere formation assay (SFA) is the gold standard assay to assess stem ...
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Virtual Screening of Transmembrane Serine Protease Inhibitors Authors:  Antti Poso, Topi Tervonen and Juha Klefström, date: 04/20/2017, view: 2462, Q&A: 0
The human family of type II transmembrane serine proteases includes 17 members. The defining features of these proteases are an N-terminal transmembrane domain and a C-terminal serine protease of the chymotrypsin (S1) fold, separated from each other by a variable stem region. Recently accumulated evidence suggests a critical role for these ...
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Cell Biology

Time-lapse Observation of Chromosomes, Cytoskeletons and Cell Organelles during Male Meiotic Divisions in Drosophila Authors:  Karin Tanabe, Ryotaro Okazaki, Kana Kaizuka and Yoshihiro H. Inoue, date: 04/20/2017, view: 2336, Q&A: 0
In this protocol, we provide an experimental procedure that perform time-lapse observation of intra-cellular structures such as chromosomes, cytoskeletons and cell organelles during meiotic cell divisions in Drosophila males. As primary spermatocyte is the largest dividing diploid cell in Drosophila, which is equivalent in size ...
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RNase Sensitivity Screening for Nuclear Bodies with RNA Scaffolds in Mammalian Cells Authors:  Taro Mannen and Tetsuro Hirose, date: 04/20/2017, view: 2363, Q&A: 0
The mammalian cell nucleus is highly organized and contains membraneless nuclear bodies (NBs) characterized by distinct resident factors. The NBs are thought to serve as sites for biogenesis and storage of certain RNA and protein factors as well as assembly of ribonucleoprotein complexes. Some NBs are formed with architectural RNAs (arcRNAs) as ...
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Reversible Cryo-arrests of Living Cells to Pause Molecular Movements for High-resolution Imaging Authors:  Jan Huebinger, Martin E. Masip, Jens Christmann, Frank Wehner and Philippe I. H. Bastiaens, date: 04/20/2017, view: 2102, Q&A: 0
Fluorescence live-cell imaging by single molecule localization microscopy (SMLM) or fluorescence lifetime imaging microscopy (FLIM) in principle allows for the spatio-temporal observation of molecular patterns in individual, living cells. However, the dynamics of molecules within cells hamper their precise observation. We present here a detailed ...
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Developmental Biology

Physical Removal of the Midbody Remnant from Polarised Epithelial Cells Using Take-Up by Suction Pressure (TUSP) Authors:  Miguel Bernabé-Rubio, David C. Gershlick and Miguel A. Alonso, date: 04/20/2017, view: 2642, Q&A: 0
In polarised epithelial cells the midbody forms at the apical cell surface during cytokinesis. Once severed, the midbody is inherited by one of the daughter cells remaining tethered to the apical plasma membrane where it participates in non-cytokinetic processes, such as primary ciliogenesis. Here, we describe a novel method to physically remove ...
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Immunology

Lentiviral Barcode Labeling and Transplantation of Fetal Liver Hematopoietic Stem and Progenitor Cells Authors:  Trine A. Kristiansen, Alexander Doyle and Joan Yuan, date: 04/20/2017, view: 2567, Q&A: 0
Cellular barcoding enables the dissection of clonal dynamics in heterogeneous cell populations through single cell lineage tracing. The labeling of hematopoietic stem and progenitor cells (HSPCs) with unique and heritable DNA barcodes, makes it possible to resolve donor cell heterogeneity in terms of differentiation potential and lineage bias at ...
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Microbiology

Isolation of the Dot/Icm Type IV Secretion System Core Complex from Legionella pneumophila for Negative Stain Electron Microscopy Studies Authors:  Tomoko Kubori and Hiroki Nagai, date: 04/20/2017, view: 1965, Q&A: 0
Legionella possesses a pivotal secretion machinery to deliver virulence factors to eukaryotic host cells. In this protocol, we describe the procedure for isolation of the native core complex of the Dot/Icm type IV secretion system from L. pneumophila aiming to perform biochemical and transmission electron microscopy analyses.
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CRISPR/Cas9 Editing of the Bacillus subtilis Genome Authors:  Peter E. Burby and Lyle A. Simmons, date: 04/20/2017, view: 4774, Q&A: 0
A fundamental procedure for most modern biologists is the genetic manipulation of the organism under study. Although many different methods for editing bacterial genomes have been used in laboratories for decades, the adaptation of CRISPR/Cas9 technology to bacterial genetics has allowed researchers to manipulate bacterial genomes with ...
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Molecular Biology

Expression and Analysis of Flow-regulated Ion Channels in Xenopus Oocytes Authors:  Shujie Shi and Marcelo D. Carattino, date: 04/20/2017, view: 2719, Q&A: 0
Mechanically-gated ion channels play key roles in mechanotransduction, a process that translates physical forces into biological signals. Epithelial and endothelial cells are exposed to laminar shear stress (LSS), a tangential force exerted by flowing fluids against the wall of vessels and epithelia. The protocol outlined herein has been used to ...
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RNA Degradation Assay Using RNA Exosome Complexes, Affinity-purified from HEK-293 Cells Authors:  Michal Domanski and John LaCava, date: 04/20/2017, view: 2463, Q&A: 0
The RNA exosome complex plays a central role in RNA processing and regulated turnover. Present both in cytoplasm and nucleus, the exosome functions through associations with ribonucleases and various adapter proteins (reviewed in [Kilchert et al., 2016]). The RNA exosome-associated EXOSC10 protein is a distributive, 3’-5’ exoribonuclease. ...
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Neuroscience

The Object Context-place-location Paradigm for Testing Spatial Memory in Mice Authors:  Edith Lesburguères, Panayiotis Tsokas, Todd Charlton Sacktor and André Antonio Fenton, date: 04/20/2017, view: 3212, Q&A: 0
This protocol was originally designed to examine long-term spatial memory in PKMζ knockout (i.e., PKMζ-null) mice (Tsokas et al., 2016). Our main goal was to test whether the ability of these animals to maintain previously acquired spatial information was sensitive to the type and complexity of the spatial information that needs ...
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Evaluation of Muscle Performance in Mice by Treadmill Exhaustion Test and Whole-limb Grip Strength Assay Authors:  Beatriz Castro and Shihuan Kuang, date: 04/20/2017, view: 3333, Q&A: 0
In vivo muscle function testing has become of great interest as primary phenotypic analysis of muscle performance. This protocol provides detailed procedures to perform the treadmill exhaustion test and the whole-limb grip strength assay, two methods commonly used in the neuromuscular research field.
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Preparation of Primary Astrocyte Culture Derived from Human Glioblastoma Multiforme Specimen Authors:  Mansoureh Hashemi and Mahmoudreza Hadjighassem, date: 04/20/2017, view: 2985, Q&A: 0
Glioblastoma multiforme (GBM) is a grade 4 astrocytoma tumor in central nervous system. Astrocytes can be isolated from human GBM. Study of astrocytes can provide insights about the formation, progression and recurrence of glioblastoma. For using isolated astrocytes, new studies can be designed in the fields of pharmacology, neuroscience and ...
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Plant Science

Forest GPP Calculation Using Sap Flow and Water Use Efficiency Measurements Authors:  Fyodor Tatarinov, Eyal Rotenberg, Dan Yakir and Tamir Klein, date: 04/20/2017, view: 2104, Q&A: 0
This is a protocol to evaluate gross primary productivity (GPP) of a forest stand based on the measurements of tree’s sap flow (SF), 13C derived water use efficiency (WUE), and meteorological (met) data. GPP was calculated from WUE and stomatal conductance (gs), the later obtained from SF up-scaled from sampled trees to stand ...
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Measurement of Stomatal Conductance in Rice Authors:  Yin Wang and Toshinori Kinoshita, date: 04/20/2017, view: 3104, Q&A: 0
Stomatal conductance, the reciprocal of stomatal resistance, represents the gas exchange ability of stomata. Generally, the stomatal conductance is higher when stomata open wider, and vice versa. In this protocol, we describe how to measure stomatal conductance in rice using Li-6400 (Licor, USA).
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Rapid Isolation of Total Protein from Arabidopsis Pollen Authors:  Ming Chang and Shanjin Huang, date: 04/20/2017, view: 2520, Q&A: 0
Arabidopsis pollen is an excellent system for answering important biological questions about the establishment and maintenance of cellular polarity and polar cell growth, because these processes are amenable to the genetic and genomic approaches that are readily available in Arabidopsis. Given that proteins are the direct ...
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Single Molecule RNA FISH in Arabidopsis Root Cells Authors:  Susan Duncan, Tjelvar S. G. Olsson, Matthew Hartley, Caroline Dean and Stefanie Rosa, date: 04/20/2017, view: 3687, Q&A: 0
Methods that allow the study of gene expression regulation are continually advancing. Here, we present an in situ hybridization protocol capable of detecting individual mRNA molecules in plant root cells, thus permitting the accurate quantification and localization of mRNA within fixed samples (Duncan et al., 2016; Rosa et al ...
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Growth Assay for the Stem Parasitic Plants of the Genus Cuscuta Authors:  Volker Hegenauer , Max Welz, Max Körner and Markus Albert, date: 04/20/2017, view: 2713, Q&A: 0
Cuscuta spp. are widespread obligate holoparasitic plants with a broad host spectrum. Rootless Cuscuta penetrates host stems with so called haustoria to form a direct connection to the host vascular tissue (Dawson et al., 1994; Lanini and Kogan, 2005; Kaiser et al., 2015). This connection allows a steady uptake ...
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Automated Tracking of Root for Confocal Time-lapse Imaging of Cellular Processes Authors:  Mehdi Doumane, Claire Lionnet, Vincent Bayle, Yvon Jaillais and Marie-Cécile Caillaud, date: 04/20/2017, view: 3072, Q&A: 0
Here we describe a protocol that enables to automatically perform time-lapse imaging of growing root tips for several hours. Plants roots expressing fluorescent proteins or stained with dyes are imaged while they grow using automatic movement of the microscope stage that compensates for root growth and allows to follow a given region of the root ...
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Stem Cell

Mimicking Angiogenesis in vitro: Three-dimensional Co-culture of Vascular Endothelial Cells and Perivascular Cells in Collagen Type I Gels Authors:  Markus Auler, Lena Pitzler, Ernst Pöschl, Zhigang Zhou and Bent Brachvogel, date: 04/20/2017, view: 4318, Q&A: 0
Angiogenesis defines the process of formation of new vascular structures form existing blood vessels, involved during development, repair processes like wound healing but also linked to pathological changes. During angiogenic processes, endothelial cells build a vascular network and recruit perivascular cells to form mature, stable vessels. ...
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